Transduced t cells expressing human sstr2 and application thereof

ABSTRACT

The present invention is directed to transduced T cells expressing at least 100,000 molecules of human somatostatin receptor 2 (SSTR2), which improves PET/CT imaging sensitivity. The present invention is also directed to transduced T cells expressing SSTR2 and chimeric antigen receptor (CAR). In one embodiment, the CAR is specific to human ICAM-1 and the CAR comprises a binding domain that is scFv of anti-human ICAM-1, or an I domain of the αL subunit of human lymphocyte function-associated antigen-1. In another embodiment, the CAR is specific to human CD19, and the CAR comprises a binding domain that is scFv of anti-human CD19. The present invention is further directed to using the above transduced T cells for monitoring T cell distribution in a patient by PET/CT imaging and/or treating cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 15/675,419, filed Aug. 11, 2017; which claims the benefit of U.S.Provisional Application Nos. 62/383,139, filed Sep. 2, 2016; and62/419,817, filed Nov. 9, 2016; which are incorporated herein byreference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grant CA178007awarded by the National Institutes of Health. The government has certainrights in the invention.

REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM

The Sequence Listing is concurrently submitted herewith with thespecification as an ASCII formatted text file via EFS-Web with a filename of Sequence Listing.txt with a creation date of Aug. 9, 2017, and asize of 10.9 kilobytes. The Sequence Listing filed via EFS-Web is partof the specification and is hereby incorporated in its entirety byreference herein.

FIELD OF THE INVENTION

The present invention relates to transduced T cells expressing humansomatostatin receptor 2 (SSTR2), and its application for monitoring Tcell distribution in a patient by PET/CT imaging. The present inventionalso relates to a method for treating cancer and monitoring CAR T celldistribution in a patient using transduced CAR T cells.

BACKGROUND OF THE INVENTION

Adoptive cell transfer (ACT) of cytotoxic T lymphocytes is being studiedas a potent treatment strategy for cancers that are refractory tostandard chemotherapy and radiation therapy. Clinical advances have beenmade in patients with metastatic melanoma using autologoustumor-infiltrating lymphocytes (TILs) and in several B-cell malignanciesusing autologous chimeric antigen receptor (CAR)-modified T cells¹.Methods used to predict or monitor the activity of infused T cells inpatients provide useful but limited data related to treatment efficacy.Current practices involve serum profiling of cytokines associated with Tcell activation, direct enumeration of tumor-specific T cell numbers inperipheral circulation, and tumor biopsies^(2,3). Changes in serumcytokine levels, while useful, likely reflect a broader, systemic immuneresponse, illustrating not only the activation of adoptively transferredT cells, but also their effects on neighboring immune cells and dyingtumor cells⁴. Similarly, while the quantification of adoptivelytransferred cells in circulation provides useful information regardingtheir proliferation, researchers and clinicians are blind as to whetherthe dynamism in T cell numbers relates to expansion at the primary tumorsite, metastatic foci, or at off-tumor sites⁵.

The imaging modalities with the highest potential for whole-bodyvisualization of cell trafficking in humans are magnetic resonanceimaging (MM), single-photon emission computed tomography (SPECT),PET/CT, or PET/MRI techniques for detection of labeled cells andcoregistration of anatomical information of the body⁸⁻¹⁰. PET (positronemission tomography) is particularly amenable to clinical use as itenables non-invasive, highly sensitive, repetitive, and quantitativeimaging of positron-emitting, target-specific probes. The introductionof microPET for small animal imaging has similarly made PET amenable topre-clinical studies¹¹. On-going activity of ACT against both on- andoff-tumor sites can therefore be monitored in vivo by quantitative,radiotracer-based imaging of T cell distribution and expansion uponinteraction with target antigen-expressing cells^(2,10,12). However,previous attempts to systemically monitor ACT in patients have yet to beadopted¹³. Passive labeling of T cells with positron emitting probes exvivo has been used to monitor the early-stage migration of infused Tcells but suffers from potential inaccuracies due to signals from deador dying cells, probe dilution upon cell division, and a limited abilityto track cells over extended periods of time due to short probehalf-life¹⁰.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D show expression of human SSTR2 by lentivirus vector in Tcells.

FIG. 1A: A schematic of the lentivirus vector encoding human SSTR2 isshown. LTR=long terminal repeat; SD=splice donor; SA=splice acceptor;EF1α=elongation factor 1α-promoter; ψ=encapsidation signal. Histogramsshow the level of SSTR2-specific antibody binding to wild-type Jurkat Tcells (top) and Jurkat T cells transduced with increasing virus titers.Percentages of SSTR2 positive cells are indicated.

FIG. 1B: Level of SSTR2-specific antibody binding to SSTR2-transducedJurkat T cells with and without pre-incubation with 1 μM octreotide (37°C., 30 min). The numbers denote mean fluorescence intensity.

FIG. 1C: DOTATOC uptake by SSTR2-transduced and wild-type Jurkat T cellsversus input DOTATOC concentration is shown. A first-order Langmuirisotherm equation was used to fit the data and to find the equilibriumdissociation constant (Kd). Confidence interval of Kd is shown inparenthesis. CPM, counts per minute.

FIG. 1D: DOTATOC uptake at 37° C. and 4° C. by SSTR2-transduced andwild-type Jurkat T cells is shown via Scatchard plot. Predicted Kd isshown. Data shown are from three independent experiments.

FIGS. 2A-2B show Structural model of LFA-1 I domain.

FIG. 2A: Schematic of LFA-1 in complex with ICAM-1. α and β chains, andmodular domains of LFA-1 integrin are labeled. Metal ions necessary forLFA-1 and ICAM-1 interaction are shown in circles.

FIG. 2B: Structural model of LFA-1 I domain and the N-terminal domain ofICAM-1 (D1) are drawn in ribbon diagram. N and C-termini, and mutationalhot spots are indicated.

FIGS. 3A-3I show quantitative PET/CT measurement of DOTATOC uptake byJurkat tumors and statistical analysis.

FIG. 3A: Tumor volume (mm³) plotted against number of days postxenograft.

FIG. 3B: Measured DOTATOC uptake is quantified as % ID/cm³ for Jurkattumors (100%-0% SSTR2+) over the course of tumor growth.

FIG. 3C: Representative PET/CT images of mice xenografted with Jurkat Tcells at 0, 0.1, 1, 10 and 100% SSTR2 expression. Images are maximumintensity projections (MIP) of the entire mouse body (˜20 mm thickplane). PET intensity is pseudo-colored in the range of 0-10% ID/cm³.

FIGS. 3D, 3G: DOTATOC uptake (% ID/cm³) for Jurkat tumors less orgreater than 65 mm³, respectively.

FIGS. 3E-3H: Simulated Gaussian distribution as a function of themeasured mean and standard deviation of DOTATOC uptake for each % SSTR2+tumor when the volume is less or greater than 65 mm³, respectively.

FIGS. 3F-3I: ROC curves of percentage sensitivity and specificity areshown for tumors <65 mm³ (C) and >65 mm³ (F). *** vs. 0%, p<0.001, **vs. 0%, p<0.01 by Student's t-test.

FIGS. 4A-4D show CAR T cell efficacy against thyroid tumor cells invitro and in vivo.

FIG. 4A: Representative histograms showing the level of anti-ICAM-1antibody binding to ICAM-1 negative HEK293T, ICAM-1 positive HeLa, and8505C cells.

FIG. 4B: Schematic of the lentivirus vector encoding SSTR2-R6.5-CAR.SS=signal sequence; TM=transmembrane; Cyt=cytoplasmic domain.Representative dot plots showing anti-SSTR2 and anti-CAR antibodybinding to non-transduced (left) and SSTR2-P2A-R6.5-CAR-transduced(right) primary human T cells.

FIG. 4C: Primary human T cells were transduced separately withindividual vectors encoding either SSTR2 or R6.5-CAR. Representativehistograms show anti-SSTR2 antibody binding to either R6.5-CAR orSSTR2-transduced (filled) and non-transduced (open) primary T cells.

FIG. 4D: E:T assay measuring lysis of target expressing (HeLa and 8505C)or control (HEK 293) cells by CAR T cells. 2.5:1 ratio of E:T was used.Percentages of live cells were measured by bioluminescence intensitynormalized to the levels of target cells incubated with non-transduced Tcells. Octreotide (1 μM) was added to SR T cells as indicated (SR+Oct).n=3-6 from three different donor T cells. NT, non-transduced.

FIGS. 5A-5D show whole body imaging of tumor growth by luminescence andDOTATOC uptake by PET/CT.

FIG. 5A: Tumor burden from the day of tumor xenograft via tail vein (X0)and 21 days post (X21) is visualized and quantified.

FIGS. 5B-5D: Transverse CT-only. PET-only, and PET/CT superimposedimages are shown with a coronal view of an entire mouse body. PET imagesare drawn in indicated ranges. Ex vivo fluorescence images of lungs andliver are shown together with flow cytometry analysis of the CD3+ (Tcell) and GFP+ (tumor cell) populations of the same lungs are also shown(FIGS. 5B-5C). Each PET/CT image is representative of at least threeindependent experiments.

FIGS. 6A-6H show quantitative PET for detection of CAR T cells andluminescence of tumor burden in survivors vs. nonsurvivors.Representative longitudinal, PET/CT (coronal view of 20 mm thick plane,MIP) and concurrent bioluminescence imaging of survivors (n=4, FIG. 6A)vs. nonsurvivors (n=5, FIG. 6E). Diaphragms are traced with dottedlines, drawn to visualize tumor burdens separately in lungs and liver.Trend-line graphs plot ROI values for DOTATOC uptake (% ID/cm³) againstbioluminescence values (photons(P)/sec) in the lungs taken from the samemice on the same day. Luminescence images are drawn with the same upperbound (10⁶ P/mm²/s) with gradually increasing lower bounds (X16, X20 for2.5×10⁴ P/mm²/s and X23, X27 for 5×10⁴ P/mm²/s) for clarity ofdelineating tumor burden. Longitudinal PET/CT images of the upper bodycropped at the kidney apex are drawn with a uniform range of DOTATOCconcentrations (0.5-5.0% ID/cm³), while whole body PET/CT images aredrawn in 0.5-5.0% ID/cm³ for FIG. 6A and 1-10% ID/cm³ for FIG. 6E.Transverse CT-only, PET-only, and PET/CT superimposed images are shownfor the livers of survivors and nonsurvivors. PET images are drawn inindicated ranges. Quantification of luminescence and DOTATOC uptake bythe lungs (FIGS. 6B, 6F) body weight change (FIGS. 6C, 6G) is shown forsurvivors and nonsurvivors. Representative longitudinal, PET/CT(transverse view of 1 mm thick plane, MIP) views of lungs are shown forsurvivors (FIG. 6D) and nonsurvivors (FIG. 6H).

FIG. 7 shows schematic of the longitudinal CAR T cell imagingexperiment. Mice were divided into four groups according to the day of Tcell treatment post tumor xenograft. SR=SSTR2-R6.5-CR T cells; SS=SSTR2,non-CAR T cells; and RR=non-SSTR2, R6.5-CAR T cells. Days of xenograft,T cell injection, imaging and post-mortem analysis are indicated.

FIGS. 8A-8D show Ex vivo analysis of CAR T cell and tumor density inlungs. Ex vivo, GFP+ tumor cell fluorescence of representative lungs andliver, and flow cytometry of total, live gated lung cells from survivors(n=4, FIG. 8A) and nonsurvivors (n=5, FIG. 8B) were shown. Organs wereharvested, imaged, and FACS analyzed on 28 days post xenograft (X28).FIG. 8C shows % CD3+ vs. DOTATOC updake. FIG. 8D shows representativehistology slides for paraffin embedded H&E stained sections of oneentire lung lobe (top), and anti-human CD3 antibody and counterstainedwith hematoxylin (middle). High magnification views (yellow dotted boxedregions in middle sections) visualize tumor cells and CD3-stained Tcells (bottom). Tumor cells are identified by dark hematoxylin stainednuclei. Human CD3+ cells are stained brown. Scale bar=2 mm for top andmiddle sections and 200 μm for bottom sections. Lungs were harvestedfrom indicated mice (X23/no T, and X28 for survivors and nonsurvivors).

FIGS. 9A-9E show longitudinal, concurrent measurements of tumor burden,T cell distribution, and cytokine release.

FIG. 9A: Schematic of third generation SSTR2-I domain-based CARconstruct.

FIG. 9B: Schematic of SSTR2-I domain based PET imaging of adoptivelytransferred CAR T cells.

FIG. 9C: Longitudinal measurements of NOTAOCT uptake by PET/CT (top halfof each panel), and tumor burden by whole body luminescence imaging(bottom half of each panel). Images are representative of four mice ineach cohort. Whole body PET/CT images, taken on the day of maximumtracer uptake, are shown on the far right. Imaging time points areindicated below the bottom panel. For example, 15 represents 15 dayspost tumor xenograft (and 7 days post T cell infusion).

FIG. 9D: Quantification of luminescence and tracer uptake in the lungsof mice treated as indicated. Top Panel: NT (non-transduced T cells.Bottom level: CARs-F292A.

FIG. 9E: Cytokine levels measured from blood drawn at various timepoints from the same mice in ‘b’ and ‘c’ are plotted (mean±SD, duplicatemeasurements). Top Panel: NT (non-transduced T cells. Bottom level:CARs-F292A.

FIGS. 10A-10B show SSTR2 application to detection of T cell distributionin the brain, spine, and bones.

FIG. 10A: Tumor-specific localization and expansion of SSTR2-CD19 CAR Tcells are confirmed by PET/CT using a radiotracer ¹⁸F-NOTA-octreotide.CAR T cell expansion is seen in the head (b-e; subarachnoid &ventricles), lungs (d-e), liver (b-e), hind limb joints (d-e), lymphnodes (c-e), and spine (e, compare with ‘a’). Radiotracer showsbackground uptake by the gall bladder (a), clearing through the gut, andkidneys and bladder (A; 20 mm MIP image). X21T14 denotes 21 days posttumor xenograft and 14 days post T cell infusion.

FIG. 10B: Bioluminescence imaging of Raji xenograft showing growth inthe brain, spine, lymph nodes, liver, and bones (front and side views).

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein, “about” refers to ±10% of the recited value.

As used herein, “adoptive T cell therapy” involves the isolation and exvivo expansion of tumor specific T cells to achieve greater number of Tcells than what could be obtained by vaccination alone. The tumorspecific T cells are then infused into patients with cancer in anattempt to give their immune system the ability to overwhelm remainingtumor via T cells which can attack and kill cancer.

As used herein, “affinity” is the strength of binding of a singlemolecule (e.g., I domain) to its ligand (e.g., ICAM-1). Affinity istypically measured and reported by the equilibrium dissociation constant(K_(D)), which is used to evaluate and rank order strengths ofbimolecular interactions.

A “binding molecular,” refers to a molecule that is capable to bindanother molecule of interest.

A “chimeric antigen receptor (CAR)” means a fused protein comprising anextracellular domain capable of binding to an antigen, a transmembranedomain derived from a polypeptide different from a polypeptide fromwhich the extracellular domain is derived, and at least oneintracellular domain. The “extracellular domain capable of binding to anantigen” means any oligopeptide or polypeptide that can bind to acertain antigen. The “intracellular domain” means any oligopeptide orpolypeptide known to function as a domain that transmits a signal tocause activation or inhibition of a biological process in a cell.

A “domain” means one region in a polypeptide which is folded into aparticular structure independently of other regions.

An “elongation factor 1 (EF-1) α promoter” is derived from the humanEEF1A1 gene that expresses the α subunit of eukaryotic elongationfactor 1. EF-1 α promoter offers a broad host range.

An “integrin” or “integrin receptor” (used interchangeably) refers toany of the many cell surface receptor proteins, also referred to asadhesion receptors which bind to extracellular matrix ligands or othercell adhesion protein ligands thereby mediating cell-cell andcell-matrix adhesion processes. Binding affinity of the integrins totheir ligands is regulated by conformational changes in the integrin.Integrins are involved in physiological processes such as, for example,embryogenesis, hemostasis, wound healing, immune response andformation/maintenance of tissue architecture. Integrin subfamiliescontain a beta-subunit combined with different alpha-subunits to formadhesion protein receptors with different specificities.

As used herein, “I domain” refers to the inserted or I domain of theα_(L) subunit of LFA-1, and is an allosteric mediator of ligand bindingto LFA-1. I domain is a native ligand of ICAM-1. The ligand binding siteof the I domain, known as a metal ion-dependent adhesion site (MIDAS),exists as two distinct conformations allosterically regulated by theC-terminal α7 helix. A wild-type (WT) I domain encompasses amino acidresidues 130-310 of the 1145 amino acid long mature α_(L) integrinsubunit protein (SEQ ID NO: 1, which is the amino acid residues 26-1170of GenBank Accession No. NP 002200). All numbering of amino acidresidues as used herein refers to the amino acid sequence of the matureα_(L) integrin (SEQ ID NO: 1), wherein residue 1 of SEQ ID NO: 1corresponds to residue 26 of the sequence of GenBank Accession No.NP_002200.

“Lymphocyte function-associated antigen-1”, “LFA-1”, “α_(L)β₂ integrin”or “CD18/CD11a” refers to a member of the leukocyte integrin subfamily.LFA-1 is found on all T-cells and also on B-cells, macrophages,neutrophils and NK cells and is involved in recruitment to the site ofinfection. It binds to ICAM-1 on antigen-presenting cells and functionsas an adhesion molecule.

As used herein, a “tumor antigen” means a biological molecule havingantigenecity, expression of which causes cancer.

A “single chain variable fragment (scFv)” means a single chainpolypeptide derived from an antibody which retains the ability to bindto an antigen. An example of the scFv includes an antibody polypeptidewhich is formed by a recombinant DNA technique and in which Fv regionsof immunoglobulin heavy chain (H chain) and light chain (L chain)fragments are linked via a spacer sequence. Various methods forpreparing an scFv are known to a person skilled in the art.

“Somatostatin receptor type 2 (SSTR2)” is a receptor for somatostatin-14and -28. Somatostatin acts at many sites to inhibit the release of manyhormones and other secretory proteins. The biologic effects ofsomatostatin are probably mediated by a family of G protein-coupledreceptors that are expressed in a tissue-specific manner. SSTR2 is amember of the superfamily of receptors having seven transmembranesegments and is expressed in highest levels in cerebrum and kidney. Afull molecule of human SSTR2 has 369 amino acids and its sequence isshown as GenBank Accession No. NP_001041. A “truncated SSTR2”, as usedherein, refers to a C-terminus shortened human SSTR2, which contains1-314 amino acid residues of human SSTR2 with a deletion of theC-terminus beyond amino acid 314⁶⁶.

A “2A peptide” is used by several families of viruses, the best knownfoot-and-mouth disease virus of the Picornaviridae family, for producingmultiple polypeptides. The mechanism by the 2A sequence for generatingtwo proteins from one transcript is by ribosome skipping—a normalpeptide bond is impaired at 2A, resulting in two discontinuous proteinfragments from one translation event. 2A peptides include porcineteschovirus-1 (P2A, SEQ ID NO: 2), equine rhinitis A virus (E2A, SEQ IDNO: 3), thosea asigna virus (T2A, SEQ ID NO: 4), or foot-and-mouthdisease virus (F2A, SEQ ID NO: 5). Table 1 shows the sequences of T2A,P2A, E2A, and F2A.

TABLE 1 Peptide Amino acid sequence T2A:     *EGRGSLLTCGDVEENPGP P2A:   *ATNFSLLKQAGDVEENPGP E2A:   *QCTNYALLKLAGDVESNPGP F2A:*VKQTLNFDLLKLAGDVESNPGP *(GSG) residues can be added to the 5′ end ofthe peptide to improve cleavage efficiency.

“A vector” is a nucleic acid molecule used as a vehicle to artificiallycarry foreign genetic material into another cell, where it can bereplicated and/or expressed. The four major types of vectors areplasmids, viral vectors, cosmids, and artificial chromosomes. A vectoris generally a DNA sequence that consists of an insert (transgene) and alarger sequence that serves as the backbone of the vector.

The inventor has discovered a method to map the physical distributionand expansion of adoptively transferred T cells throughout the body in alongitudinal manner with transduced T cells having sufficient surfacereporter molecules to provide high sensitivity of PET/CT imaging. Themethod significantly improves real-time monitoring of T cell activityagainst tumor, reduces potential toxicity from off-tumor site targeting,and contributes to exploring adjuvant therapies to enhance adoptive Tcell efficacy against solid cancers.

The present invention is directed to transduced T cells that efficientlyexpress surface reporters, e.g., sodium iodide symporter (NIS)¹⁷,prostate-specific membrane antigen (PSMA)¹⁸, or human somatostatinreceptor 2 (SSTR2)¹⁹. In one embodiment, the present invention isdirected to transduced T cell expressing at least 100,000 molecules ofthe surface reporter per T cell, or at least 300,000 molecules per Tcells, or at least 600,000 molecules per T cells; preferably 1 millionmolecules per T cell. The high level of expression of surface reportermolecules improves PET/CT imaging sensitivity, i.e., improves a minimumnumber of T cells that can be detected by labelled NIS, PSMA, SSTR2 onthe surface of the T cells. The site density of the expression of thesurface reporter molecules on T cells, for example, can be determined byincubating non-transduced and SSTR-transduced T cells withDOTATOC/DOTATATE and then measuring the DOTATOC/DOTATATE uptake. Thevalues obtained are used for Scatchard analysis to determine theaffinity (K_(D)) and site density (surface reporter molecules per Tcell). For imaging purpose in the present invention, a full molecule ofhuman SSTR2 (access number NP_001041) or a truncated SSTR2, containing1-314 amino acid residues of the full molecule of human SSTR2 can beused.

In one embodiment, the T cell has been transduced with a lentivirusvector that expresses a human surface reporter such as human SSTR2,PSMA, or NIS. The present invention provides a lentivirus vectorcomprising an elongation factor 1a promoter and a nucleic acid moleculeencoding SSTR2, PSMA, or NIS; the lentivirus vector comprises the humanSSTR2 gene, PSMA gene, or NIS gene operably linked to (e.g., downstreamof) the elongation factor-1α promoter, as illustrated in FIG. 1A. TheEF-1 alpha promoter, which offers a broad host range, is derived fromthe human EEF1A1 gene that expresses the alpha subunit of eukaryoticelongation factor 1.

The present invention is directed to a method for monitoring T celldistribution in a patient. The method can be used in adoptive T celltherapy, or in hematopoietic stem cell transplant. In a firstembodiment, the surface reporter (e.g. SSTR2) is pre-labelled in vitro.The first method comprises the steps of: incubating transduced T cellsthat efficiently express a surface reporter with a radioactive labelthat binds to the surface reporter, intravenously infusing the labelledT cells into a patient, and detecting the labelled T cell distributionby PET/CT imaging. In one embodiment, the labelled T cells administeredto the patient are in an amount of 10⁴-10⁸, or 10⁶-10⁸, or 10⁶-10⁷cells/kg of the patient.

In a second embodiment, the surface reporter (e.g. SSTR2) is labelledpost-infusion in vivo. The second method comprises the steps of:intravenously infusing the transduced T cells into a patient, injectingto the patient a radioactive label that binds to the surface reporter atleast one hour prior to PCT/CT imaging, and detecting the labelled Tcell distribution by PET/CT imaging. In one embodiment, the transduced Tcells administered to the patient are in an amount of 10⁴-10⁸, or10⁶-10⁸, or 10⁶-10⁷ cells/kg of the patient.

The present invention provides stable transduction of T cells with aspecific reporter gene, which allows for extended longitudinal studiesusing serial infusions of reporter-specific probes. Additionally, asonly live cells are capable of continually expressing the reporter gene,observed signals are limited to these cells only.

SSTR2 is a preferred surface reporter for the present invention forseveral reasons. One is to take advantage of the FDA-approved PETradiotracer DOTATOC or DOTATATE, which is currently in use in clinics toprobe for overexpressed SSTR2 in neuroendocrine tumors⁴³. SPECT-basedimaging is also available using ¹¹¹In-DTPAOC (Octreoscan)⁴⁴. SSTR2displays restricted basal expression in tissues and all major organsexcept in the kidneys and cerebrum making it ideal for detection ofadoptively transferred T cells targeting a multitude of solid tumors.SSTR2 can potentially function as a dual reporter-suicide gene byconjugation of the therapeutic high-energy radioisotopes ¹⁷⁷Lutetium,⁹⁰Yttrium, or ²¹³Bismuth to DOTATOC instead of ⁶⁸Gallium^(25,45), thusenabling elimination of SSTR2 expressing T cells in the case of CARtoxicity. SSTR2 is surface expressed and therefore does not requireprior radioligand internalization into the cell. It has previously beenshown that SSTR2 facilitates rapid radiotracer uptake and this combinedwith swift renal clearance of unbound DOTATOC means that high quality,clinical-grade images can be obtained at one hour post DOTATOCinjection⁴⁶. DOTATOC also has a short half-life of 68 min which,combined with its rapid clearance, delivers a low radiation dose to thepatient. The fact that SSTR2 is of human origin should also limit itsimmunogenicity which has plagued experiments using non-human geneticreporters^(48,49).

The inventor has discovered that co-expression of CAR with separatepopulation-specific reporter genes, for example SSTR2 and PSMA, followedby sequential, time-delayed injection of cognate PET radiotracers,reveals dynamics of, and interactions between, these populations in bothclinical and pre-clinical studies. The present invention is directed totransduced T cells that efficiently express surface receptors, andchimeric antigen receptor (CAR). In one embodiment, the transduced Tcells express at least 100,000 molecules, or at least 300,000 molecules,or at least 600,000 molecules, or at least 1 million molecules, ofreporters (e.g., SSTR2 or PSMA) per T cell. In one embodiment, the CARis specific to an intracellular adhesion molecule-1 (ICAM-1), which isoverexpressed in a range of malignant cancers²⁷⁻³² such as thyroidcancer, gastric cancer, pancreatic cancer, or breast cancer. In anotherembodiment, the CAR is specific to a tumor antigen such as CD19.

In another aspect of the invention, the inventor has designed a singlelentivirus vector to engineer human primary T cells to express both ahuman cell surface reporter (e.g. SSTR2, PSMA or NIS) and a CAR, whichis specific to ICAM-1 or CD19.

The present invention provides a lentivirus vector encoding human SSTR2and chimeric antigen receptor (CAR) specific to human ICAM-1 or humanCD19. In one embodiment, the lentivirus vector further encodes an aminoacid cleavage sequence C-terminal to the human SSTR2 or the CAR, whereinthe amino acid cleavage sequence comprises a 2A peptide from porcineteschovirus-1 (P2A), equine rhinitis A virus (E2A), thosea asigna virus(T2A), or foot-and-mouth disease virus (F2A).

In the lentivirus vector of the present invention, the CAR comprises (i)a binding domain to human ICAM-1 or human CD19, (ii) a transmembranedomain, (iii) at least one co-stimulating domain, and (iv) an activatingdomain. The binding domain to human ICAM-1 in CAR is scFv of anti-humanICAM-1, or an I domain of the αL subunit of human lymphocytefunction-associated antigen-1. The binding domain to human CD19 in CARis scFv of anti-human CD19.

In one embodiment, the CAR of the present invention comprises (i) ahuman I domain that binds specifically to ICAM-1. The I domain may be awild type I domain, or a mutant thereof having 1 to 3 mutations. Idomain specific to ICAM-1 can be built using the I domain derived fromLFA-1 (FIGS. 2A and 2B). Various activating point mutations in the Idomain are localized outside of the binding interface that includes aregion known as the metal-ion dependent adhesion site (MIDAS) (FIG. 2B).Mutants containing the step-wise elevation of I domain affinity toICAM-1 from 1 mM to 1 nM can be obtained by screening a library ofmutants for their higher binding to ICAM-1 coated surface, beads, orcells. For example, different affinity mutants can be isolated using ayeast display system (see Jin et al.⁶²). Affinity is first measured bysurface plasmon resonance (e.g., Biacore) to assess 1:1 binding affinitybetween I domain and ICAM-1. Affinity of ICAM-1 to CAR expressed oncells can be measured by flow cytometry and using the Langmuir isothermequation. Likewise, Scatchard analysis can be performed to estimate CARaffinity by measuring the amounts of free and cell-surface bound ligand(in this case, radio- or fluorescence-labeled ICAM-1).

Table 2 shows measured affinities of LFA-1 I domains of wild type andmutants to ICAM-1. A majority of mutations are changing hydrophobicbulky side chains (F, L, I) into more hydrophilic (A, S, T), therebydisrupting the structure of more compact, low affinity I domainconformation. For example, substitution of Phe-292 located in theC-terminal α7-helix with Ala (F292A) and Gly (F292G) provides toaffinities (K_(D)) of ˜20 μM and 0.1 respectively (Table 2). Thecombination of F292G with another comparably activating mutation inPhe-265 (F265S/F292G) provides an affinity of 6 nM, approximately200,000-fold higher than the wild-type (WT) I domain (K_(D)=1.5 mM)(FIG. 1C). To lock the C-terminal α7-helix of F265S/F292G in the openposition (FIG. 1A), Gly-311 can be replaced with Cys (G311C) in theF265S/F292G mutant (F265S/F292G/G311C, dubbed triple mutant or TM) toform a disulfide bond with the naturally unpaired Cys-125 (Table 2).Therefore, the monovalent affinities of individual I domain variants forICAM-1 can be designed to span approximately six orders of magnitude(K_(D)˜1 nM to 1 mM), as measured by surface plasmon resonance (SPR) orestimated by flow cytometry (FIG. 1C, Table 2). The mutants in Table 2are for illustration purpose only; the CARs of the present invention arenot limited to these specific mutants. Mutants that have other mutationsand have affinities to ICAM-1 between 1 mM to 1 nM can be made, tested,and selected according to methods known to a skilled person.

TABLE 2 Sequence of Name SEQ ID NO: 1 Affinity Wild-type (WT) G128-G3111.5 mM* I288N G128-G311 202 μM** I309T G128-G311 127 μM** L295AG128-G311 37 μM** F292A G128-G311 20 μM* F292S G128-G311 1.24 μM** L289GG128-G311 196 nM** F292G G128-G311 119 nM* F265S G128-G311 145 nM*F265S/F292G (DM) G128-G311 6 nM* F265S/F292G/G311C (TM) E124-S313 ~1 nM*R6.5 scFv 10 nM*** *SPR measurements; **Estimated from flow cytometrymean fluorescence intensity (MFI) values of ICAM-1-Fc binding to yeastcells expressing I domain variants³. The equation used was Kd (M) =0.00175*exp(−0.1542*MFI); ***Estimated from titrated R65 antibodybinding to HeLa cells³⁴.

In one embodiment, the CAR of the present invention comprises I domainthat is a wild-type human I domain, a mutant of wild-type human I domainhaving 1 to 3 amino acid mutations, or a sequence having at least 95%,or at least 96% identity, or at least 97% identity, or at least 98%identity, or at least 99% identity to the sequence of the wild-type Idomain or the mutant, having an affinity of binding human ICAM-1 of 1 mMor stronger. In one embodiment, the mutant may have one or moremutations at the amino acid residue 265, 288, 289, 292, 295, 309, or 311of the wild-type I domain. For example, the mutant may have one or moremutations of I288N, I309T, L295A, F292A, F292S, L289G, F292G, F265S,F265S/F292G, or F265S/F292G/G311C, of the wild-type I domain. Ingeneral, combining two I domain mutations produces a mutant with ahigher affinity than that of each parent mutant. For example, combiningtwo mutants each having about 100 μM Kd typically produces a mutanthaving about 1 to about 10 μM Kd range. F292G is a very potent pointmutation; combining F292G with other mutations increases I domainaffinity to ICAM-1 to stronger than 100 nM Kd. The above numbering ofthe amino acid residues is in reference to the mature amino acidsequence of SEQ ID NO: 1, and residue number 1 corresponds to the aminoacid residue 26 of GenBank Accession No. NP_002200.

In one embodiment, the CAR of the present invention comprises I domainthat binds ICAM-1 at an affinity between 1 mM to 1 nM Kd, preferably1-200 μM Kd or 1-20 μM Kd.

In one embodiment, the CAR of the present invention comprises I domainthat binds to ICAM-1 at an affinity between about 120 nM to about 1 nMKd, e.g., F292G, F265S, F265S/F292G, and F265S/F292G/G311C.

In one embodiment, the CAR of the present invention comprises I domainthat binds to ICAM-1 at an affinity between about 20 μM to about 120 nMKd, e.g., F292A, F292S, and 1289G.

In one embodiment, the CAR of the present invention comprises I domainthat binds to ICAM-1 at an affinity between about 200 μM to about 20 μMKd, e.g., I288N, I309T, L295A, and F292A.

In one embodiment, the CAR of the present invention comprises I domainthat binds to ICAM-1 at an affinity between about 1 μM to about 100 μMKd, e.g., L296A, F292A and F292S.

In one embodiment, the CAR of the present invention comprises I domainthat binds to ICAM-1 at an affinity between about 1 mM to about 200 μMKd, e.g., wild-type and I288N.

In one embodiment, the CAR of the present invention comprises I domainthat binds to ICAM-1 at an affinity between about 1 mM to about 100 μMKd, e.g., wild-type, I288N, and I309T. The affinities in the aboveembodiments refer to the interaction between I domain and ICAM-1 insolution.

The CAR of the present invention comprises (ii) a transmembrane domainwhich spans the membrane. The transmembrane domain may be derived from anatural polypeptide, or may be artificially designed. The transmembranedomain derived from a natural polypeptide can be obtained from anymembrane-binding or transmembrane protein. For example, a transmembranedomain of a T cell receptor α or β chain, a CD3 zeta chain, CD28,CD3-epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64,CD80, CD86, CD134, CD137, ICOS, CD154, or a GITR can be used. Theartificially designed transmembrane domain is a polypeptide mainlycomprising hydrophobic residues such as leucine and valine. In preferredembodiments, the transmembrane domain is derived from CD28 or CD8, whichgive good receptor stability.

The CAR of the present invention comprises (iii) one or moreco-stimulatory domains selected from the group consisting of human CD28,4-1BB (CD137), ICOS-1, CD27, OX 40 (CD137), DAP10, and GITR (AITR). Inembodiment, the CAR is a third generation and comprises twoco-stimulating domains such as CD28 and 4-1BB.

The endodomain (the activating domain) is the signal-transmissionportion of the CAR. After antigen recognition, receptors cluster and asignal is transmitted to the cell. The most commonly used endodomaincomponent is that of CD3-zeta (CD3 Z or CD3ζ), which contains 3 ITAMs.This transmits an activation signal to the T cell after antigen isbound. CD3-zeta may not provide a fully competent activation signal andadditional co-stimulatory signaling may be needed. For example, one ormore co-stimulating domains can be used with CD3-Zeta to transmit aproliferative/survival signal.

The CAR of the present invention may comprise a signal peptideN-terminal to the I domain so that when the CAR is expressed inside acell, such as a T-cell, the nascent protein is directed to theendoplasmic reticulum and subsequently to the cell surface, where it isexpressed. The core of the signal peptide may contain a long stretch ofhydrophobic amino acids that has a tendency to form a singlealpha-helix. The signal peptide may begin with a short positivelycharged stretch of amino acids, which helps to enforce proper topologyof the polypeptide during translocation. At the end of the signalpeptide there is typically a stretch of amino acids that is recognizedand cleaved by signal peptidase. Signal peptidase may cleave eitherduring or after completion of translocation to generate a free signalpeptide and a mature protein. The free signal peptides are then digestedby specific proteases. As an example, the signal peptide may derive fromhuman CD8 or GM-CSF, or a variant thereof having 1 or 2 amino acidmutations provided that the signal peptide still functions to cause cellsurface expression of the CAR.

The CAR of the present invention may comprise a spacer sequence as ahinge to connect I domain with the transmembrane domain and spatiallyseparate antigen binding domain from the endodomain. A flexible spacerallows to the binding domain to orient in different directions to enableits binding to a tumor antigen. The spacer sequence may, for example,comprise an IgG1 Fc region, an IgG1 hinge or a CD8 stalk, or acombination thereof. A human CD28 or CD8 stalk is preferred.

In one embodiment, the lentivirus vector includes a third generation ofCAR, and the vector encodes a fusion protein comprising human SSTR2,porcine teschovirus-1 2A, a binding domain (scFv of anti-human ICAM-1,scFv of anti-human CD19, or human I domain), transmembrane domain ofCD28, cytoplasmic domain of CD28, CD137, and CD 3ζ, from N-terminus toC-terminus (see FIG. 4B). In another embodiment, the lentivirus vectorincludes a second generation of CAR. The vector encodes a fusion proteincomprising human SSTR2, porcine teschovirus-1 2A, a binding domain (scFvof anti-human ICAM-1, scFv of anti-human CD19, or human I domain),transmembrane domain of CD28, cytoplasmic domain of CD28, and CD 3ζ fromN-terminus to C-terminus. Alternatively, the vector encodes a fusionprotein comprising human SSTR2, porcine teschovirus-1 2A, scFv ofanti-human ICAM-1, transmembrane domain of CD8, cytoplasmic domain ofCD137, and CD 3ζ, from N-terminus to C-terminus.

The present invention provides a nucleic acid encoding the CAR describedabove. The nucleic acid encoding the CAR can be prepared from an aminoacid sequence of the specified CAR by a conventional method. A basesequence encoding an amino acid sequence can be obtained from theaforementioned NCBI RefSeq IDs or accession numbers of GenBenk for anamino acid sequence of each domain, and the nucleic acid of the presentinvention can be prepared using a standard molecular biological and/orchemical procedure. For example, based on the base sequence, a nucleicacid can be synthesized, and the nucleic acid of the present inventioncan be prepared by combining DNA fragments which are obtained from acDNA library using a polymerase chain reaction (PCR).

The nucleic acid encoding the CAR of the present invention can beinserted into a vector, and the vector can be introduced into a cell.For example, a virus vector such as a retrovirus vector (including anoncoretrovirus vector, a lentivirus vector, and a pseudo type vector),an adenovirus vector, an adeno-associated virus (AAV) vector, a simianvirus vector, a vaccinia virus vector or a Sendai virus vector, anEpstein-Barr virus (EBV) vector, and a HSV vector can be used. As thevirus vector, a virus vector lacking the replicating ability so as notto self-replicate in an infected cell is preferably used.

For example, when a retrovirus vector is used, the process of thepresent invention can be carried out by selecting a suitable packagingcell based on a LTR sequence and a packaging signal sequence possessedby the vector and preparing a retrovirus particle using the packagingcell. Examples of the packaging cell include PG13 (ATCC CRL-10686),PA317 (ATCC CRL-9078), GP+E-86 and GP+envAm-12, and Psi-Crip. Aretrovirus particle can also be prepared using a 293 cell or a 293T cellhaving high transfection efficiency. Many kinds of retrovirus vectorsproduced based on retroviruses and packaging cells that can be used forpackaging of the retrovirus vectors are widely commercially availablefrom many companies.

The present invention provides T cells modified to express a reportermolecule (e.g., SSTR2, PMSA, or NIS) and the CAR as described above.CAR-T cells of the present invention bind to ICAM-1 or CD19 viaanti-ICAM-1 antibody, or I domain, or anti-CD19 antibody of CAR, therebya signal is transmitted into the cell, and as a result, the cell isactivated. The activation of the cell expressing the CAR is varieddepending on the kind of a host cell and an intracellular domain of theCAR, and can be confirmed based on, for example, release of a cytokine,improvement of a cell proliferation rate, change in a cell surfacemolecule, killing target cells, or the like as an index.

T cells modified to express SSTR2 and CAR can be used as a therapeuticagent for a disease. The therapeutic agent comprises the T cellsexpressing the I domain-CAR as an active ingredient, and may furthercomprise a suitable excipient. Examples of the excipient includepharmaceutically acceptable excipients known to a person skilled in theart.

The invention is further directed to a method for treating cancer andmonitoring CAR T cell distribution in a patient. The method comprisesthe steps of: incubating the transduced CAR T cells with a radioactivelabel that binds to SSTR2, intravenously infusing the labelled CAR Tcells into a patient, detecting the labelled CAR T cell distribution byPET/CT imaging, and infiltrating the labelled CAR T cells into cancercells to kill cancer cells. In this method, SSTR2 is pre-labelled invitro. In one embodiment, the labelled CAR T cells is administered in anamount of 10⁴-10⁸, or 10⁶-10⁸, or 10⁶-10⁷ cells/kg of the patient.

In an alternative method, the method comprises the steps of:intravenously infusing the transduced CAR T cells into a patient,injecting to the patient a radioactive label that binds to SSTR2 atleast one hour prior to PCT/CT imaging, detecting the labelled CAR Tcell distribution by PET/CT imaging, and infiltrating the labelled CAR Tcells into cancer cells to kill cancer cells. In this method, SSTR2 islabelled post-infusion in vivo. In one embodiment, the transduced CAR Tcells is administered in an amount of 10⁴-10⁸, or 10⁶-10⁸, or 10⁶-10⁷cells/kg of the patient.

Adoptively transferred T cells have been shown to penetrate anddistribute throughout tumor tissue¹². The utility of any imagingmodality capable of monitoring ACT therefore depends upon alimit-of-detection threshold sensitive enough to enable monitoring ofmeaningful T cell activity at low tissue densities. Moreover, evaluationof this detection limit in a physiologically relevant model must alsoaccount for the dynamic sensitivity and specificity of the infusedimaging agent for the reporter in question, whose density at the tumorsite will vary exponentially as its cognate T cells expand and contractin response to target interaction. Therefore, to approximate theDOTATOC-based detection limit of tumor infiltrating SSTR2-transduced Tcells, the inventor utilized mosaic tumor xenografts of Jurkat T cellswith increasing ratios of SSTR2 expression to create a standard by whichquantitative PET signals can be related to T cells of known densitywithin tumors. Using the lentivirus developed in this study, the surfaceexpression of SSTR2 in transduced Jurkat T cells was in the range ofseveral million per cell, a level significantly higher than previouslypublished¹⁹ (36,000 copies per Jurkat T cell) and therefore extendingthe lower-limit of SSTR2+ T cell detection.

The inventor observed that with a known threshold of radiotracer uptake,one can detect tumor-infiltrating T cells down to a minimum density of0.8% or ˜4×10⁶ cells/cm³, with 95% specificity and 87% sensitivity. Thiscompares favorably to a previous report using PET to detecttumor-infiltrating T cells that used flow cytometry-based detection in aseparate, equally treated cohort of mice as a reference to purport atumor-infiltrating T cell detection limit of 0.5% within tumors⁵³.

To demonstrate the feasibility of SSTR2 reporter-based imaging topredict and monitor tumor-directed T cell activity, the inventor choseICAM-1 positive anaplastic thyroid cancer cells as a target model andengineered T cells to express both ICAM-1-specific CAR and SSTR2 using asingle lentiviral vector. A potential drawback to the use of geneticreporters for imaging receptor-modified ACT is that the T cells inquestion require the stable expression of two separate genes, which cansubstantially reduce the percentage of cells co-expressing both genes.However, with the use of a self-cleaving ‘P2A’ sequence³⁹, the inventordemonstrated that both CAR and reporter genes can be successfullyexpressed on the same individual cell without compromising the level ofexpression otherwise achievable using independent vectors. T cellsexpressing the R6.5 CAR efficiently and specifically lysed ICAM-1expressing 8505C and HeLa tumor cell lines, as monitored bybioluminescence, within 24 hr whilst leaving ICAM-1 negative HEK293cells largely untouched. Furthermore, efficient killing was alsoobserved at lower E:T ratios, thus demonstrating both specificity andhigh activity of the R6.5 CAR for ICAM-1 expressing target cells. Thistargeting efficacy was replicated in vivo as treatment of 8505Ctumor-bearing mice with SSTR2-R6.5-CAR T cells resulted in a significantreduction in tumor burden across all treated mice compared to those thatreceived T cells expressing SSTR2 only. Tumor lysis occurred 1-2 weekspost treatment with the time required for CAR mediated tumor reductioncorrelating with burden at time of treatment. Expanding SSTR2-R6.5-CAR Tcells at the tumor site were visualized by increasing DOTATOC signals.PET imaging of SSTR2-R6.5-CAR T cells was sensitive enough to visualizetheir perfusion throughout the lungs such that the lung footprint andoutline became distinguishable by the presence of T cells alone. Thiswas confirmed by histological analysis of tissue sections, whichdemonstrated the ubiquitous presence of human CD3+ T cells throughoutthe lungs of treated mice. Despite the small size (several mm) ofmetastatic nodules in the liver, localized DOTATOC accumulation was alsorecorded which coincided with the emergence, followed by attenuation, ofdistinct liver tumor nodules as detected by bioluminescence imaging.Extrapolation of the image quality and sensitivity obtained in thecurrent study to similar scenarios in humans would likely enable equalif not better monitoring of CAR tracking to primary, metastatic andcritically, to on-target, off-tumor-sites. Visualization of CARexpansion may even draw attention to previously undetected metastaticsites. Therefore, sufficiently sensitive reporter imaging of ACT mayprovide additional prognostic capabilities.

Due to reported dose-limiting toxicity, the number of modified T cellsinfused to patients is typically in the range of 1-10×10⁶ Tcells/kg^(2,3,41,54) and target-mediated expansion and persistence of Tcells is therefore a prerequisite for substantive tumor destruction tooccur. Indeed, it has been reported that higher peak expansion ofinfused T cells correlates with increased rates of diseaseremission^(2,54). Tumor-bearing mice in the current study were treatedwith approximately 1.5×10⁶ SSTR2-CAR+ T cells at days 7-15 postxenograft. Subsequent longitudinal monitoring of both tumor growth andCAR expansion at the tumor site enabled several observations to be made.Timely infusion of CAR T cells resulted in survival of all subjectswithout any weight loss, while a later treatment led to uniform deathdespite the evidence of tumor killing by CAR T cells.

Survivors exhibited a biphasic pattern of DOTATOC uptake within thelungs, with a similar luminescence pattern observed regarding primarylung tumors. A notable exception to this correlative pattern was thatpeak DOTATOC signal, and therefore peak T cell expansion, lagged behindpeak tumor burden by several days. This expansion of T cells past theonset of target elimination may result from enduring cognate antigenmediated signals causing continued CAR T cell expansion before eventualexhaustion and contraction occurred⁵⁵. Swift contraction of CAR T cellsfollowing peak expansion indicates that target antigen density hasfallen to levels no longer capable of sustaining CAR expansion and thattumor elimination was achieved without immediate relapse. The biphasicpattern of DOTATOC uptake in survivors stood in stark contrast to theunrelenting increases in both T cell and tumor burdens in non-survivorswhere tumor growth was evidently surpassing the rate of killing by Tcells. It would be interesting to investigate whether a similar patternis observed in additional tumor models and in clinical studies. Finally,DOTATOC uptake values obtained in the longitudinal study enabledcomparisons with uptake values derived from the SSTR2-titrated Jurkatmodel. This indicated that peak CAR T cell density in 8505C tumorsranged from below 1% at infusion to ˜10% in survivors and to well over10% in mice with high tumor burden.

In summary, the inventor utilized a genetic reporter, somatostatinreceptor 2 (SSTR2) and positron emission tomography (PET) toquantitatively and longitudinally visualize whole body T celldistribution and anti-tumor dynamics. SSTR2-based PET was applied to anACT model using chimeric antigen receptor (CAR) T cells specific tointercellular adhesion molecule-1 that is overexpressed in anaplasticthyroid tumors. Timely CAR T cell infusions resulted in the survival ofall subjects bearing rapidly growing tumors, while later T cellinfusions led to uniform death. Quantitative, longitudinal PET imagingof T cells revealed a biphasic expansion and contraction response amongsurvivors, with peak tumor burden preceding peak T cell burden byseveral days. In contrast, non-survivors displayed unrelenting increasesof both tumor burden and T cell number, indicating that the rate oftumor growth was outpacing that of T cell killing. The inventordemonstrates that the prognostic utility of SSTR2-based longitudinalimaging, directly relating biphasic CAR T cell actions to tumorelimination, may apply to close monitoring of ACT efficacy and overallresponse in patients.

The inventor has demonstrated a clinically adaptable, quantitativeimaging system capable of specifically detecting adoptively transferredCAR T cells and monitoring their target-specific expansion andcontraction at the tumor site with unprecedented detail. A simple methodfor estimating the density of solid tumor-infiltrating T cells has alsobeen established. The inventor anticipates that the SSTR2 system can beeasily adapted to multiple ACT models and can facilitate efforts towardsincreasing our understanding of the parameters behind the success andfailures of ACT with particular regard to monitoring systemic toxicitiesand the responses to solid tumors.

The following examples further illustrate the present invention. Theseexamples are intended merely to be illustrative of the present inventionand are not to be construed as being limiting.

EXAMPLES Materials and Methods Example 1. Mammalian Cell Culture

Parental HeLa, HEK 293 (ATCC), and 8505C (DSMZ, Germany) cells weretransduced with lentivirus encoding Firefly Luciferase-F2A-GFP(Biosetta) followed by fluorescence activated cell sorting (FACS) topurify GFP expressing cells. HeLa-FLuc⁺GFP⁺ and HEK 293-FLuc⁺GFP⁺ cellswere cultured in Advanced Dulbecco's Modified Eagle Medium containing10% (v/v) fetal bovine serum (FBS), 2 mM L-alanyl-L-glutamine dipeptide(Gibco), and 100 U/ml Penicillin-Streptomycin (Pen/Strep) (Gibco).8505C-FLuc⁺GFP⁺ cells were cultured in RPMI-1640 supplemented with 10%(v/v) FBS, 2 mM L-alanyl-L-glutamine dipeptide, and 100 U/ml Pen/Strep.Human peripheral blood was obtained from healthy volunteer donors byvenipuncture. This protocol is approved by an Institutional Review Boardof Weill Cornell Medicine (Permit Number: #1302013613). Peripheral bloodmononuclear cells (PBMC) were isolated over Ficoll-Paque PLUS (GEHealthcare) and cultured in Optimizer CTS T-cell Expansion SFM (Thermo)supplemented with 5% human AB serum (Sigma), 2 mM L-alanyl-L-glutaminedipeptide, 100 U/ml Pen/Strep and 30 IU/ml human IL-2 (Cell Sciences).Non-adherent cells were removed after 24 hr and magnetically enrichedfor T cells with Dynabeads CD3/CD28 T cell expander (Thermo) at a 2:1bead:T cell ratio. Dynabead-bound T cells were subsequently cultured inIL-2 containing media at a density of 1-2×10⁶ cells/ml. All cells wereincubated at 37° C. in a 5% CO₂ humidified incubator.

Example 2. Construction of ICAM-1 CAR and SSTR2 Reporter Genes

The CAR gene specific to ICAM-1 was derived from the scFv sequence of amurine monoclonal anti-human R6.5 antibody^(56,57)—itself derived fromhybridoma (ATCC). The R6.5 specific scFv was then fused to thetransmembrane and cytoplasmic domains of CD28, CD137, and CD3ζ of anindependent third generation pLenti plasmid (a kind gift from Dr. CarlJune at PENN³⁵). A lentivirus vector (derived from CAR vector) encodinghuman SSTR2 (NM_001050) was constructed by synthesizing SSTR2 codingsequencing (IDT) and inserting it into the vector using Xba1 and Sal1sites.

Example 3. Construction of I Domain CAR and SSTR2 Reporter Genes

Genetic sequences encoding LFA-1 I domains of varying affinities toICAM-1 were derived from a previous study⁶². I domain variants werefused at the C-terminus directly to the CD8 hinge, CD28 transmembranedomain, and the intracellular portions of the 3^(rd) generation CARarchitecture incorporating the cytoplasmic domains of CD28, CD137, andCD3ζ. The complete CAR inserts were then subcloned into a pLentibackbone³⁵. A reporter gene for CAR T cell imaging, SSTR2, was linked toI domain at the N-terminus using a ‘ribosome skipping’ porcineteschovirus-1 2A (P2A) sequence to ensure comparable production of CARand SSTR2 from the same mRNA.

Example 4. Lentivirus Production and Transduction of T Cells

Lentivirus particles were produced by transiently transfecting HEK 293cells using calcium phosphate. Briefly, 10 μg transfer gene, 7.5 μgCMV-dR8.2 (Addgene) and 5 pCMV-VSVG (Addgene) were mixed and incubatedwith 2 M CaCl₂ followed by 2×HBSS. Resulting solutions were addeddropwise to 10 cm² cell culture dishes seeded with 3.2×10⁶ HEK 293 in 10ml DMEM 24 hr previously. Transfection media was replaced after 6 hr.Media containing lentivirus was harvested at 48 and 72 hr posttransfection, filtered through 0.45 μm filters and concentrated byultracentrifugation at 75,000×g for 2 hr at 4° C. Lentivirus was thenresuspended in serum containing media at an approximate titer of 10⁸/mland frozen at −80° C. Human T cells were transduced 24-72 hr postactivation with CD3/CD28 Dynabeads either by spinfection at 1,000× g for1 hr at 32° C. or by overnight incubation of lentivirus in the presenceof Synperonic F108 (Sigma)⁵⁸. T cells were also transduced a secondtime, 24 hr after initial transduction. The virus titer was adjusted toobtain a transduction level of approximately 50%. During and followingtransduction, media containing IL-2 was replaced with media containinghuman IL-7 (10 ng/ml) and IL-15 (5 ng/ml) (Peprotech) which was found toaugment T cell persistence in vitro^(59,60) Jurkat T cells weretransduced by a single incubation with lentivirus overnight in thepresence of Synperonic F108.

Example 5. Confirmation of SSTR2 Functionality and Measurement of SiteDensity

SSTR2-transduced Jurkat T cells were incubated with or withoutoctreotide, 1 μM (Sigma) for 30 min at 37° C. Subsequent internalizationof SSTR2 was measured by flow cytometry analysis of SSTR2 expression.The site density of SSTR2 expression on Jurkats was determined byincubating non-transduced and SSTR2-transduced Jurkat T cells withDOTATOC (250 nM-8 nM) at either 37° C. or 4° C. for 30 min in PBS/0.1%BSA. After incubation, cells were washed three times and DOTATOC uptakewas measured using a gamma counter (Packard, Cobra II Auto—Gamma).Values obtained were used for Scatchard analysis to estimate affinityand site density.

Example 6. Subcutaneous Jurkat T Cell Xenograft

All animal experiments were performed in strict accordance with therecommendations in the Guide for the Care and Use of Laboratory Animalsof the National Institutes of Health. This study's animal protocol wasapproved by the Institutional Laboratory Animal Use and Care Committeeof Weill Cornell Medicine. SSTR2 expressing Jurkats were spiked withincreasing numbers of non-transduced Jurkats to derive distinct culturescontaining defined percentages of SSTR2 expression ranging from 100-0%.For each subcutaneous xenograft, 5×10⁶ total cells were resuspended in100 μl Matrigel Basement Membrane Matrix (Corning) and injectedbilaterally into nonobese diabetic (NOD)/LtSz Prkdc^(scid)Il2rg^(tm1Wjl)/J (NSG) mice (Jackson Laboratory). Measurements of tumorsize were made using an external digital caliper. Tumor volume wascalculated by using the modified ellipsoid formula ½(Length×Width).Length was measured as the longer dimension. Each dimensionalmeasurement was rounded to the nearest 0.5 cm.

Example 7. Labeling of ⁶⁸Ga-DOTATOC

DOTATOC(1,4,7,10-tetraazacyclododecane-N^(I),N^(II),N^(III),N^(IIII)-tetraaceticacid (D)-Phe¹-Tyr³-octreotide, GMP grade) was obtained as a 1 mglyophilized powder (ABX Pharmaceuticals). The DOTATOC vial content wasdiluted with 18 MW water to 2 ml (0.5 mg/ml solution) and stored at 4°C. as a stock solution. ⁶⁸Ga was obtained by eluting an ITG ⁶⁸Ge/⁶⁸Gagenerator (ITM) with 4 ml 0.05M HCl solution. To the eluted ⁶⁸Ga³⁺, 50μl of the DOTATOC stock solution (25 μg) was added, followed by 80 μl ofa 3 N NaOAc solution for buffering. The mixture was immediately placedin a Thermomixer (Eppendorf) at 95° C. and incubated for 15 minutes.Following incubation the mixture was passed through a previouslyactivated C-18 Sep-Pak Lite (Waters) to trap the labeled peptide. Thelabeling vial was washed with 5 ml 18 MW water and the resulting washwas also passed through the C-18 Sep-Pak. Finally, the Sep-Pak waswashed with an extra 5 ml of 18 MW water to eliminate any remaining free⁶⁸Ga. The trapped ⁶⁸Ga-DOTATOC was then slowly eluted from the C-18Sep-Pak using 100 μl of ethanol followed by 900 μl of saline solutionfor injection, providing the final product in a 10% EtOH isotonic,injectable solution. The purity of the final product was checked byreverse phase HPLC.

Example 8. E:T Assay

2×10⁵HeLa-FLuc⁺GFP⁺, 8505c-FLuc⁺GFP⁺ or HEK 293-FLuc+GFP+ cells wereco-cultured with either non-transduced or CAR expressing T cells(SSTR2-R6.5 or R6.5) at varying E:T ratios as indicated. Co-cultureswere carried out in ‘T cell media’ containing 150 μg/ml D-Luciferin(Gold Biotechnology) with no cytokine supplementation. Luminescence wasmeasured using a plate reader (TECAN infinite M1000 PRO) with readingsin each E:T condition normalized to the non-transduced T cell:targetco-culture controls.

Example 9. 8505C Mouse Model, Measurement of Ex Vivo Organs andWhole-Body Tumor Growth

1×10⁶ 8505c-FLuc⁺GFP⁺ cells were injected into NSG mice via tail vein.2-3×10⁶ primary human T cells were injected via tail vein 7-15 daysafter tumor cell injection. Luminescence imaging of tumor xenografts inlive mice was performed using a whole body optical imager (In-VivoExtreme 4MP, Bruker). Mice were first anesthetized with 3% isoflurane at2 L/min O₂ and subsequent to this, maintained at 2% isoflurane at 2L/min O₂. Growth or reduction in tumor burden was estimated byintegration of luminescence over the lungs or the entire mouse body.Ex-vivo fluorescence imaging of mouse liver, lungs, spleen and resectedtumors were performed using a whole body optical imager (In-Vivo F Pro,Bruker).

Example 10. PET/CT Imaging

Registered CT images were acquired using a micro-PET/CT scanner (Inveon,Siemens) at 1-2 hr post DOTATOC injection. Projection data was acquiredin a cone-beam geometry with approximately 1s steps at 1 degree angularincrements. At least 10 million coincidence events were acquired for PETper study using a 250 to 750 keV energy window and a 6 ns timing window.A reference was included using a tube containing 100 μl of 10% ID/cm³for quantification of DOTATOC uptake in vivo. To compute DOTATOC uptakeby Jurkat tumors, the ellipsoidal ROIs (Amide) were placed to enclosesubcutaneous tumors that closely match overall tumor size and shape. Forsystemic 8505C tumor models, ellipsoids were drawn separately on theleft and right sides of lungs to enclose much of five lobes of mouselungs. The % ID/cm³ values, computed relative to the counts in areference tube, can be approximated to a standard uptake value (SUV⁶¹)by dividing % ID/cm³ by four, assuming injection efficiency of 100% and25 g of body weight. Visualization and analyses of PET/CT images wereperformed using Amide (amide.sourceforge.net).

Example 11. Flow Cytometry

Jurkat tumor xenografts or mouse organs (lungs and liver) were harvestedfrom mice following completion of PET/CT imaging. Tissues were diced andflushed through an 80 μm cell strainer to yield single cell suspensions.Red blood cells were lysed by incubating cell suspension with 1×Ammonium-Chloride-Potassium lysing buffer (ThermoFisher), followed bywashing and re-suspension in 1× HBSS containing 2% normal goat serum.Prior to staining, cells were blocked with mouse IgG at 2 μg/ml for 10min. This was followed by live staining with 1 μg/ml Propidium Iodide(Invitrogen) in combination with 2 μg/ml murine anti-human CD3-AlexaFluor 647 (Biolegend) or 2 μg/ml PE-conjugated murine anti-human SSTR2(Clone #402038, R&D). Flow cytometry gates were determined first basedon live cell gating (Propidium Iodide negative) and subsequently bystaining of respective antibodies. ICAM-1 expression on tumor cell lineswas determined using a murine anti-human R6.5 monoclonal antibody (10μg/ml) derived from hybridoma (ATCC)⁵⁷. R6.5-CAR expression on T cellswas detected using FITC-conjugated goat anti-mouse F(ab′)2 secondaryantibody (Thermo).

Example 12. Histology

Jurkat tumor xenografts were harvested, fixed in 4% paraformaldehyde inPBS, embedded in paraffin, and were cut to produce 5 μm sections(Microtome, Leica). Paraffin embedded sections were stained withhematoxylin and eosin (H&E) or hematoxylin only for CD3 immunostaining(performed by HistoWiz, Inc.). After euthanasia, mouse lungs wereperfused via trachea with 4% paraformaldehyde, and each of five lobeswas separated post fixation and embedded in paraffin. Liver tissue washarvested and processed identically for histology. Histological analysiswas performed by an experienced pathologist.

Results Example 13. Expression and Characterization of SSTR2 in Jurkat TCells

We constructed a lentivirus vector for expression of SSTR2 by insertingthe human SSTR2 gene downstream of the elongation factor-1α promoter³⁵.With increasing lentivirus titer, 100% of Jurkat T cells were transducedto express SSTR2 as measured by antibody binding (FIG. 1A). Consistentwith agonist-induced internalization of SSTR2³⁶, incubation of T cellswith the synthetic SSTR2 agonist octreotide, reduced surface expressionof SSTR2 as indicated by reduced antibody binding (FIG. 1B). Labeling ofSSTR2-transduced Jurkat T cells with ⁶⁸Ga-DOTATOC (hereafter referred toas DOTATOC) at concentrations ranging from 250 nM to 8 nM followed afirst-order Langmuir isotherm equation, giving a dissociation constant(Kd) of 38 nM (FIG. 1C). In comparison, DOTATOC uptake by non-transducedT cells was approximately 10-fold lower. Similar to the Kd estimated byLangmuir isotherm, Scatchard analysis estimated the Kd of DOTATOC to be32 nM and the site density of SSTR2 to be approximately 3.2×10⁶molecules per cell (FIG. 1D). Actual site density is likely to be lowerdue to recycling of SSTR2 after internalization of DOTATOC and somelevel of non-specific binding. Incubation of cells at 4° C. to inhibitSSTR2 recycling resulted in an estimated site density of 1.8×10⁶molecules per cell; however, the affinity of DOTATOC for SSTR2 was alsodetermined to be substantially lower (Kd=170 nM) at this temperature(FIG. 1D).

Example 14. PET Imaging of DOTATOC Uptake in SSTR2+ T Cell Xenografts

To examine the utility of SSTR2 for the detection of sparselydistributed T cells in tumors, we produced subcutaneous Jurkat T cellxenografts in mice with a mixture of SSTR2-transduced (referred to asSSTR2+) and non-transduced (wild-type) cells. These were titratedagainst each other immediately prior to xenografting to vary the levelsof SSTR2 expressing cells within tumors from 0% to approximately 100%.Xenografted Jurkat T cell tumors began to show palpable growth at ˜30days post xenograft and exhibited continuous growth for the next 25days, reaching approximately 0.7 cm³ (FIG. 3A). Longitudinal measurementof tumor size across different SSTR2+ tumors revealed that the level ofSSTR2 expression had no significant effect on the growth of Jurkat Tcell tumors. We tested our ability to detect SSTR2 positive T cellswithin tumors when both their tumor density and absolute numbers rangedfrom low to high in both respects. Thus to cover this range, weinitiated PET/CT imaging at 12 days post xenograft of 0% to 100% SSTR2+tumors, that is, before we could detect palpable tumor growth, andcontinued imaging until 56 days post xenograft (FIG. 3B). The level ofDOTATOC uptake by tumors was quantified as percent injection dose pervolume (% ID/cm³) based on the region of interest (ROI) enclosingtumors. The ROIs were defined by tumor-size measurements and anatomicalinformation from CT images. Over the course of tumor growth, DOTATOCuptake was higher in tumors comprising increasing percentages of SSTR2+cells (FIG. 3B). PET/CT images showed DOTATOC uptake by tumors anduniformly higher uptake by the kidneys and bladder—consistent with itsknown biodistribution and renal clearance (FIG. 3C)¹⁹. DOTATOC uptakevalues, as measured by % ID/cm³, agreed with visual assessments ofPET/CT images over the course of tumor growth and correlated withincreasing SSTR2+ percentages within the tumors (FIGS. 3B-3C). We alsonoted a minor increase in DOTATOC uptake during growth of 0% SSTR2+tumors, which we speculate to be caused by the increasing leakyvasculature and stagnant blood pooling within tumors (FIG. 3B).

Example 15. Defining Detection Limit, Specificity, and Sensitivity ofSSTR2+ T Cells

We next analyzed DOTATOC uptake values to determine the detectionsensitivity and specificity of SSTR2-expressing T cell density withintumors. Regarding small, palpable tumors (below ˜65 mm³), DOTATOC uptakewas slightly higher in those containing 1% SSTR2+ cells (0.64±0.4%ID/cm³; p=0.1 vs. 0% SSTR2+), but was significantly higher in tumorscontaining SSTR2+ T cell densities of 10% (0.83±0.5% ID/cm³) and 100%(3.4±1.7% ID/cm³), compared to uptake within 0% SSTR2+ tumors (0.44±0.2%ID/cm³) (FIG. 3D). Assuming a normal distribution of DOTATOC uptakevalues (FIG. 3E), a threshold of 0.6% ID/cm³ was the DOTATOC uptakecutoff in order to obtain 95% specificity (5% false positive rate) fortumors of this size. The detection sensitivity (% true positive rates)for 1, 10, and 100% SSTR2+ tumors was calculated to be 54%, 69%, and95%, respectively. The detection limit for 10% SSTR2+ tumors below ˜65mm³ was considered marginally acceptable with an area under the receiveroperating characteristic (ROC) curve (AUC)³⁷ value of 0.75 (FIG. 3F). Incontrast, DOTATOC uptake for more discernible SSTR2+ tumors (larger than65 mm³) was significantly higher within 100, 10, and 1% SSTR2+ tumors(7.1±2.3, 2.8±1.1, and 2.0±0.77% ID/cm³, respectively) when compared tobackground uptake by 0% SSTR2+ tumors (0.8±0.35% ID/cm³) (FIG. 3G).Uptake was also detectable, although not significant, even within 0.1%SSTR2+ tumors (1.1±0.51% ID/cm³, p=0.12). A DOTATOC uptake cutoff of1.1% ID/cm³ gave 95% specificity and 87% sensitivity for 1% SSTR2+tumors within the Jurkat model (FIG. 3H). With the same threshold, oneachieves >95% sensitivity for tumors where the SSTR2+ T cell density isat or above 10%. The ROC curve of DOTATOC uptake by 1% SSTR2+ tumorgives 0.95 AUC, while 0.1% SSTR2+ tumor gives 0.68 AUC (FIG. 3I). SSTR2+T cells in culture were ˜100% SSTR2 positive by antibody staining.However, later resection of 100% SSTR2+ tumor followed by staining forSSTR2 by flow cytometry, revealed that the level of SSTR2 expression wasreduced to ˜80%, with the 20% reduction reflecting the presence of mousestroma cells within tumors. The reduction of SSTR2 expression was notcaused by the loss of SSTR2 expression during tumor growth as a similarlevel of reduction was seen by CD3 staining: 70% positive in cellsharvested from tumors, reduced from 80-85% CD3 positive from the samecells in culture.

To express the observed SSTR2+ T cell tumor density as an absolutenumber per volume, we resected Jurkat tumors and determined the averagetotal cell density to be 5.1±1.3×10⁸/cm³ (n=4). Accordingly, 1% SSTR2+tumors (comprising approximately 4×10⁸/cm³ Jurkat T cells and 1×10⁸/cm³stroma) would contain ˜4×10⁶ SSTR2 positive T cells per cm³. Based onthe data obtained using the Jurkat model, we proceeded to utilizeSSTR2-based PET imaging to estimate and longitudinally monitor thedensity of reporter-expressing CAR T cells infiltrating solid tumors.

One-way ANOVA and unpaired Student's t-test were performed using Prism 7(GraphPad) on data indicated.

Example 16. Efficacy of ICAM-1 Specific CAR T Cells Against ThyroidTumor Cells

Radioiodine resistant, poorly differentiated thyroid cancers have beenfound to overexpress ICAM-1 at levels correlating with tumor malignancyand metastatic potential³⁰. The anaplastic thyroid cancer cell line8505C was also found to be ICAM-1 positive (FIG. 4A), the level of whichvaried due to culture conditions and growth in vivo. The anti-ICAM-1 CARis 3^(rd) generation and consists of an ICAM-1-specific single-chainvariable fragment (scFv) from the R6.5 antibody³⁸, the transmembrane andcytoplasmic domains of CD28, followed by CD137 and CD3ζ³⁵. A lentiviralvector encoding both SSTR2 and R6.5-CAR was constructed by linking SSTR2to the R6.5-CAR with a ‘ribosome skipping’ porcine teschovirus-1 2A(P2A) sequence³⁹ (FIG. 4B). Primary T cells were transduced to expressSSTR2 and R6.5-CAR at approximately 50% (FIG. 4C). Expression levels ofboth SSTR2 and R6.5-CAR obtained via the single SSTR2-R6.5-CAR (SR)vector were comparable to what could be attained by separate SSTR2 (SS)and R6.5-CAR (RR) vectors at similar virus titers (FIG. 4C). To test theselectivity of CART cell-mediated killing of 8505C cells, we also usedthe cervical cancer cell line, HeLa, exhibiting high basal levels ofICAM-1, and the ICAM-1 negative cell line, HEK 293 as positive andnegative controls, respectively. All target cells were lentivirallytransduced to express GFP and firefly luciferase in order toquantitatively monitor cell viability. Incubation of CAR T cells withICAM-1 positive and negative cell lines showed that CAR T cell killingof target was strictly dependent upon ICAM-1 expression. After 24 hours,17±11% of HeLa and 52±15% of 8505C cells were viable at effector totarget (E:T) ratios of 2.5:1 with no killing of HEK 293 cells observed(106±8%) (FIG. 4D). Additionally, no killing of 8505C cells (97±2%) wasobserved upon coincubation with SS T cells. The rate of killing of 8505Ccells by RR and SR T cells, and that of HeLa by SR T cells with orwithout the addition of octreotide (1 μM) was comparable. This confirmedthat SSTR2 expression on T cells, as well as binding of its cognateligand (DOTATOC), does not alter CAR T cell functionality.

Example 17. Longitudinal PET Imaging of SSTR2+ CAR T Cells In Vivo

In order to test the SSTR2-based reporter system's ability to acquire invivo visual mapping of CAR T cell localization and anti-tumor dynamics,NSG mice were first xenografted by systemic injection of 1×10⁶ 8505C-FLuc⁺GFP⁺ cells. Bioluminescence imaging demonstrated that primary8505C tumors localized to the lungs with metastases occurring within theliver and at more distant sites (FIG. 5A), consistent with previouslyreported observations of 8505C tumor growth characteristics in mice⁴⁰.Using bioluminescence as a measure of tumor burden, it was observed thatan initial infusion of 1×10⁶ tumor cells could expand 50-fold to anestimated 50×10⁶ cells over 21 days, corresponding to an in vivo celldoubling time of ˜4 days. Tumor growth was by not hampered by treatmentwith SS T cells 10-13 days post-tumor xenograft, confirming the absenceof non-specific killing by non-CAR T cells in vivo. Prior to conductingDOTATOC imaging of SR T cells, we first examined the circumstances thatgive rise to non-specific DOTATOC uptake, which may include leaky tumorvessles within areas of high tumor burden causing blood pooling. First,CT images of mice with no tumors that were infused with SS T cellsshowed the heart and tumor-free alveolar air space in the lungs (FIG.5B). The level of DOTATOC uptake in these mice (0.7% ID/cm³) wascomparable to those in Jurkat tumors containing 0% SSTR2+ cells(0.6-0.8% ID/cm³) and is thus indicative of the absence of CAR T cellexpansion. In comparison, mice xenografted with 8505C tumors hadalveolar air space replaced by the growth of tumor cells (accounting foras much as 22% of total live cells in lungs), which generated increasedalveolar tissue density in transverse CT images of these mice (FIG. 5C).We observed low yet gradually increasing non-specific DOTATOC uptake inthe thoracic cavity of mice with high tumor burden (1.3±0.5% ID/cm³ for23-27 days post-tumor xenograft) that received either no T cells or SS Tcells. This was found to be due to subjects' poor health and slowerheartbeat, causing a longer circulation time and a delay in DOTATOCclearance. Mice treated with RR T cells (RR 1-3, FIG. 7) were also foundto show only background levels of DOTATOC uptake in the lungs (1.0%ID/cm³). This was despite the fact that over the course of tumorkilling, RR T cells had expanded such that they accounted for 36% oftotal live cells in lungs.

After confirming the relative absence of lung-specific DOTATOC uptakerelated to tumor burden itself or SSTR2-negative T cell expansion, wethen treated tumor-bearing mice with SR T cells at day 7, 10, 13, and 15post xenograft. Mice were subjected to longitudinal bioluminescence andconcurrent PET/CT imaging to visualize the relationship between tumorburden and the dynamism of infused SR T cell numbers over the course oftheir localization, tumor engagement, and killing (FIG. 6A, 6E, and FIG.7). Progressive delays in T cell infusion post tumor xenograft were usedin order to observe potential differences in CAR T cell dynamics relatedto differences in tumor burden. Primary tumor burden in the lungs frommice treated with SR T cells 7 and 10 days post-xenograft (SR1-4) begannoticeably decreasing at approximately 16 days post tumor-xenograft withlittle change in body weight (FIG. 6C). Increasing tumor burden overtime correlated with increasing DOTATOC uptake within the lungsindicating that infused SR T cells had stably localized to the tumorsite where they were actively proliferating in response to engagementwith ICAM-1-positive tumor cells (FIG. 6A). It was also found that peakDOTATOC uptake, and therefore peak CAR T cell expansion, lagged behindpeak tumor bioluminescence by approximately 4 days. Peak DOTATOC uptakewithin the lungs was followed by gradual curtailment and contraction ofDOTATOC signal corresponding to diminishing T cell numbers. In contrastto the swift tumor elimination, followed by CAR T cell contractionwithin the lungs of surviving mice, further delay of treatment with SR Tcells to 13-15 days post tumor-xenograft (SR5-9) failed to reduce tumorburden and restore health before euthanasia was invoked (deemednecessary after a 25-30% loss in body weight) (FIGS. 6C, 6G). This wasdespite increasing lung-specific DOTATOC uptake confirming SR T cellexpansion in the lungs of these mice. Longitudinal transverse CT imagesrevealed that while the density in lungs associated with tumor and CAR Tcell burden persisted in nonsurvivors, it was eventually eliminated andreverted to clear, normal alveolar density upon tumor elimination andcontraction of T cells in surviving mice (FIGS. 6D, 6H).

Example 18. Confirmation of PET and Bioluminescence Imaging by Ex VivoAnalysis

Assessment of CAR T cell-mediated tumor killing by ex vivo fluorescenceimaging of the lungs and liver (performed on day 28 post-tumorxenograft) confirmed that tumor elimination had indeed occurred insurvivors treated with SR T cells on 7-10 days post tumor xenograft(FIG. 8A). Flow cytometry analysis of lungs that were determined to haveundergone CAR T cell contraction by DOTATOC uptake, showed that CD3levels were reduced to less than 1% of total lung cells (FIG. 8A). Italso confirmed continuing expansion of SR T cells and substantialreduction of tumor burden in nonsurviving mice treated on days 13-15post tumor xenograft (2.5%), compared to mice that received no T cells(22%) (FIG. 8B vs. FIG. 5C). Histological analysis of lung tissuesfurther corroborated the conclusions drawn from whole bodyPET/bioluminescence imaging, flow cytometry, and ex vivo organ images.Lung tissues from untreated mice (X23/no T cells) revealed extensiveinfiltration and growth of tumor cells into the alveoli, bronchioles,pulmonary vessels, and pleura spaces compared to their healthy ‘notumor’ counterparts (FIG. 8D). Lung tissues harvested from survivors(SR1-4) were mostly devoid of tumor infiltrates and SR T cells, andappeared to have restored normal alveoli structure (FIG. 8D).Nonsurvivor lung tissues (SR5-9) revealed much higher burden of tumor,yet infiltrating T cells (CD3 staining) were found to co-localize withtumor lesions, confirming on-going tumor elimination by CAR T cells(FIG. 8D).

In contrast to the more evenly distributed and synchronized growth oftumors in the lungs, metastatic lesions in the liver were distinctive,isolated and smaller, measuring only several mm in size (FIGS. 5C-5D,FIGS. 8A-8B). In most cases, these liver metastases would be ofinsufficient size to generate CAR expansion to requisite densities forvisual detection by DOTATOC/PET—even during efficient CAR T cellresponses. However, we were able to detect elevated DOTATOC levels incertain livers with liver tumor metastases as visualized byluminescence, particularly within nonsurvivors (FIG. 8B). Such elevatedDOTATOC accumulation was scarce in survivors where large tumor lesionsin the liver were absent (FIG. 8A). Infiltration of CAR T cells intometastatic liver lesions irrespective of their size was also confirmedby histology. This data therefore confirmed that dynamic DOTATOC uptakeby SSTR2-expressing, adoptively transferred T cells can be used tomonitor the presence of T cell infiltrates at both primary tumor sitesand metastatic lesions.

Example 19. Real-Time Imaging of CAR T Cell Kinetics, Efficacy, andToxicity

To spatiotemporally monitor T cell distribution in real-time by PET/CT,we introduced an imaging reporter gene, SSTR2 into the I domain CARvector using a ribosome skipping P2A sequence to ensure equal expressionof CAR and the reporter on the surface of T cells (FIG. 9A). FIG. 9Bshows schematic of SSTR2-I domain based PET imaging of adaptivetransferred CAR T cells. Expression of SSTR2 enabled binding andintracellular accumulation of an infused, positron-emitting,SSTR2-specific radiotracer, ¹⁸F-NOTA-Octreotide. Emitted signals werethen detected with high resolution with no tissue penetration issues bya micro PET scanner. Flow cytometry measurements of SSTR2 reporter geneand Myc-tag expression representing CAR on the surface of primary humanT cells. Expression of SSTR2 and Myc tagged I domain was confirmed byantibody staining by flow cytometry measurements of SSTR2 reporter geneand Myc-tag expression representing CAR on the surface of primary humanT cells.

Mice were xenografted with 8505C tumors as before, and were treated withNT or F292A CAR T cells. Whole-body luminescence imaging was performedto estimate tumor burden while PET/CT imaging was performed on the sameday to track CAR T cell distribution (FIG. 9B). At each time point,blood was collected to measure human cytokines for correlation with Tcell dynamics. PET/CT images in mice displayed expected backgroundlevels in gall bladder, kidneys and bladder caused by radiotracerexcretion (FIG. 9C; far-right). In the NT treated control cohort, asmall but gradual increase in non-specific tracer uptake was observed,which was due to increasing tumor burden and the associated increase inblood pooling (FIG. 9C). In contrast, specific tracer uptake wasobserved in mice treated with SSTR2-F292A CAR T cells, demonstrating theexpansion and contraction phases in the lungs, with peak CAR T cellsignal occurring approximately at 22 days post xenograft, which is 4days following peak tumor burden (18 days post xenograft), and graduallydecreasing to background levels (FIGS. 9C-9D). This shows biphasic Tcell expansion and contraction phenomenon.

Cytokine analysis of serum obtained from treated mice demonstrated asurge in IFN-γ, IL-6, and CXCL10 concentrations prior to peak T cellexpansion, which also returned to background levels post tumorelimination and following contraction of T cell density in the lungs tobackground levels (FIG. 9E).

Example 20. Real-Time Imaging of CD19-CAR T Cells

To demonstrate broad applicability of SSTR2-based imaging of CAR Tcells, we used Burkitt lymphoma (Raji) xenograft, which is one of thetumor model being used by others for validation of CD19-specific CAR Tcells^(63,64). Similar to findings of ICAM-1 overexpression inhematological cancer (including multiple myeloma⁶⁵), we foundoverexpression of ICAM-1 (˜3×10⁵/cell) in Raji cells. Our studyconfirmed the characteristics of Raji tumor growth, mainly appearing inthe central nervous system (subarachnoid space, brain, and spine),liver, bones, and lymph nodes (FIG. 10B). Due to the tumor growth andcompaction of spinal nerve, mice rapidly develop hind limb paralysis,followed by total paralysis and death. Therapeutic efficacy of CAR Tcells was easily validated by either prevention of paralysis or reversalof partial hind limb paralysis, in addition to tumor killing assessed bybioluminescence imaging. PET/CT imaging of mice with ¹⁸F-NOTA-octreotiderevealed our ability to detect CAR T cell localization and expansionthroughout the body, which overlapped with tumor bioluminescence (FIG.10A). Our data therefore proved that our imaging technique is applicableto CAR T cell imaging at the brain, spine, lungs, liver, bone, and lymphnodes.

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It is to be understood that the foregoing describes preferredembodiments of the present invention and that modifications may be madetherein without departing from the scope of the present invention as setforth in the claims.

What is claimed is:
 1. Transduced T cells that express SSTR2 andchimeric antigen receptor (CAR) specific to ICAM-1.
 2. The transducedCAR T cells of claim 1, wherein the CAR comprises the binding domain tohuman ICAM-1, and the binding domain is scFv of anti-human ICAM-1, or anI domain of the αL subunit of human lymphocyte function-associatedantigen-1.
 3. The transduced CAR T cells of claim 2, wherein the bindingdomain to human ICAM-1 is a wild type I domain having the sequence of130-310 amino acids of SEQ ID NO: 1, a mutant having 1 to 3 mutations ofthe wild type, or a sequence having at least 95% identity to the wildtype.
 4. The transduced CAR T cells of claim 3, wherein the bindingdomain to human ICAM-1 is a wild type I domain having the sequence of130-310 amino acids of SEQ ID NO:
 1. 5. The transduced CAR T cells ofclaim 3, wherein the binding domain to human ICAM-1 is a mutant having 1to 3 mutations of the wild type I domain having the sequence of 130-310amino acids of SEQ ID NO:
 1. 6. The transduced CAR T cells of claim 5,wherein the mutant comprises a mutation of I288N, I309T, L295A, F292A,F292S, L289G, F292G, or F265S, of the wild-type I domain having thesequence of 130-310 amino acids of SEQ ID NO:
 1. 7. The transduced CAR Tcells of claim 5, wherein the mutant comprises two mutations of F265Sand F292G, of the wild-type I domain having the sequence of 130-310amino acids of SEQ ID NO:
 1. 8. The transduced CAR T cells of claim 5,wherein the mutant comprises three mutations of F265S, F292G, and G311C,of the wild-type I domain.
 9. The transduced CAR T cells of claim 2,wherein the binding domain to human ICAM-1 is scFv of anti-human ICAM-1.10. The transduced CAR T cells of claim 2, wherein the CAR furthercomprises a transmembrane domain, at least one co-stimulating domain,and an activating domain.
 11. The isolated nucleic acid according toclaim 10, wherein the co-stimulatory domain is selected from the groupconsisting of CD28, 4-1BB, ICOS-1, CD27, OX-40, GITR, and DAP10.
 12. Theisolated nucleic acid according to claim 10, wherein the activatingdomain is CD3 zeta.
 13. The transduced CAR T cells of claim 1, whichexpresses at least 100,000 molecules of SSTR2 per T cell.
 14. A methodfor treating cancer and monitoring CAR T cell distribution in a patient,comprising the steps of: incubating the transduced CAR T cells of claim1 with a radioactive label that binds to SSTR2, intravenously infusingthe labelled CAR T cells into a patient in an amount of 10⁶-10⁸ cells/kgpatient, and detecting the labelled CAR T cell distribution by PET/CTimaging, whereby the labelled CAR T cells are infiltrated into cancercells to kill the cancer cells.
 15. The method according to claim 14,wherein the cancer is thyroid cancer, gastric cancer, pancreatic cancer,or breast cancer.
 16. The method according to claim 14, wherein theradioactive label is ⁶⁸Ga-DOTATOC or ⁶⁸Ga-DODATATE.
 17. A method fortreating cancer and monitoring CAR T cell distribution in a patient,comprising the steps of: intravenously infusing the transduced CAR Tcells of claim 1 into a patient, injecting to the patient a radioactivelabel that binds to SSTR2 at least one hour prior to PCT/CT imaging, anddetecting the labelled CAR T cell distribution by PET/CT imaging,whereby the labelled CAR T cells are infiltrated into cancer cells tokill the cancer cells.
 18. The method according to claim 17, wherein thecancer is thyroid cancer, gastric cancer, pancreatic cancer, or breastcancer.
 19. The method according to claim 17, wherein the radioactivelabel is ⁶⁸Ga-DOTATOC or ⁶⁸Ga-DODATATE.